SK14792003A3 - EF-TU binding agent as antibacterial agent - Google Patents

Abstract

The invention relates to the use of substances, binding to the bacterial translation factor EF-Tu, to prevent the formation of a cytoskeleton in bacterial cells and for production of anti-bacterial agents. The invention further relates to anti-bacterial agents, comprising partial sections of the amino acid sequences of the domains 2 and/or 3 of a bacterial EF-Tu protein, preferably with a length of 4-20 amino acids.

Description

Use of EF-Tu-bound substances, an antibacterial agent containing EF-Tu-bound substances, and a method for identifying new antibacterial active agents

Technical field

The invention relates to the use of substances bound to the bacterial translation factor EF-Tu to prevent the formation of a cytoskeleton in bacterial cells and to produce antibacterial agents. The invention further relates to antibacterial compositions comprising partial stretches of amino acid sequences of domains 2 and / or 3 of a bacterial EF-Tu protein, preferably having a length of 4 to 20 amino acids.

BACKGROUND OF THE INVENTION

Penicillin or other antibiotics have been used inter alia as antibacterial agents, which have a specific effect in preventing bacterial cell growth. This effect is that these antibiotics prevent the necessary widening of peptidoglycan bonds in cell growth. Growing cells are decisively weakened by this destabilization of mureins. The bacteria in the stationary phase do not stop because there is no expansion of the murein bonds at this stage.

The bacterial EF-Tu protein comprises domains 1, 2 and 3 (Song, H., Parsons, M.R., Rowell, S., Leonard, G., Philips, E. V .: J Mol Biol, 285, 1245-1256, 1999). The sequences of the EF-Tu protein and its coding genes for Escherichia coli and for a number of other bacteria are published and available in databases. It is also described that EF-Tu domain 1 plays a role in protein synthesis.

The possible presence of prokaryotic persistent cytoskeletons has been described in Naturwissensch., 85, 1998, 278-282 (Mayera et al.).

However, the involvement of EF-Tu bacterial proteins in the production of such cytoskeleton was unknown.

From literature to date (eg Schilstra, M.J., Slot, JW, van der Meide, P.H., Posthuma, G., Cremers, A.F., Bosch, L .: Immunochemical Localization of Elongation

-2 factor Tu in Escherichia coli., Biochem. Biophys. Acta 1291, (1996), 122130) with respect to the arrangement of EF-Tu in bacterial cells, it follows that EF-Tu is virtually homogeneously distributed in the cytoplasm. It has not been taken into account in the prior art that artificially produced EF-Tu fibrils can be cold-depolymerized in vitro.

Surprisingly, it has been found that there is a cytoskeleton in prokaryotic cells that can be stained with anti-EF-Tu antibodies. This cytoskeleton comprises a network of protein fibrils that are located near the surfaces of the cytoplasmic membranes directed to the cytoplasm and are dispersed throughout the cytoplasm. The cytoplasmic membrane and the peripheral part of this network system can be considered as 2 concentric empty tubes, the cytoplasmic membrane representing the outer part of both tubes, the peripheral part of the network (cytoskeleton) representing the inner tube. The fibrils passing through the cytoplasm complete and stabilize the system and are the site of ribosome deployment, the ribosomes could also be detected in the peripheral part of the cytoskeleton oriented towards the cytoplasm.

Prokaryotic cytoskeleton represents several variants:

- variants that mediate special functions consist of proteins that are close to higher cells and, in rod-like bacteria, determine the length and diameter of the cells,

- variants that make up proteins that are close to tubulin of higher cells and take care of the ordered cell distribution, as well as

- one basic variant occurring in all prokaryotes ("basic cytoskeleton"), consisting of a network system of protofilaments of the EFT protein (elongation factor Tu), which serves as a form of stabilizing structural component to the cell and acts as a attachment component for ribosomes and other complex molecular molecules clusters. The latter variant is also referred to herein as the cytoskeletal network.

EF-Tu is a protein that contains 3 domains, wherein Domain 1 participates in the translation process. To date, no specific function has been described for domains 2 and 3. It has now been found that the exposed epitopes of domains 2 and 3 form a fitting, with the surface of one being convex and the surface of the other being concave. It is believed that these fits can affect the formation of EF-Tu polymers, especially linearly arranged fibrils, both in vitro and in vivo. These fibrils are components of a network system that acts as a cytoskeleton. Maybe from that

It will be appreciated that substances that bind to EF-Tu, particularly in the domain of domains 2 or 3, can serve to prevent the formation of cytoskeleton in bacterial cells and can be used to produce an antibacterial composition.

The cytoskeletal network can thus be considered a target (target) for a new class of antibiotics.

In particular, EF-Tu may serve as a target protein for novel antibacterial agents that can occupy fusions on Domain 2 and / or 3, thereby preventing the formation of EF-Tu polymers in the cell, which are essential for bacterial cell structure. This mode of action is fundamentally different from the mode of action of other EF-Tu acting antibiotics (e.g., Vogeley, L., Palm, GJ, Mesters, JR, Hilgenfeld, R .: Conformational change of the elongation factor Tu (EF-Tu) induced by binding anibiotics J Biol Chem, 276 (2001), 17149-17155 It is reported that hitherto known chiromycin-type antibiotics act to prevent the reversibility of one conformational end of domain 1, with domain 1 bending in the direction of domain 2 when GTP binds This mechanism is fundamentally different from the polymerization braking effect described herein, in which domains 2 and 3 are involved.

EF-Tu comprises 394 amino acids. Domain 1 includes amino acids 8 to 204, with amino acids 172 to 204 forming the binding structure to domain 2. Domain 2 includes amino acids 205 to 298 and domain 3 includes amino acids 299 to 394.

There are different secondary structures within domains 2 and 3. Of particular importance are amino acid sequences 317 to 328 and 343 to 354, which are located in domain 3 and form bows that are free in space and are candidate sequences for altered activity by amino acid sequences that are located at the corresponding ending at the edge of domain 2, amino acid sequences range from 218 to 224.

According to the invention, it has surprisingly been found that damage to bacterial cytoskeleton cells can be achieved by preventing EFT polymerization. Such cell damage is mainly achieved in conventional bacterial cells having a cell wall. The invention is particularly applicable to eubacteria.

Substances that can be used to inhibit the formation of cytoskeleton may be of a different nature as long as they are able to block the effect of the exchange between domain 2

-4a domain 3 of two adjacent EF-Tu molecules. Suitable substances may be e.g. identify by a procedure that includes:

(a) contacting one of the test substances with a bacterial EF-Tu or a fragment thereof that is capable of polymerizing, e.g. with a fragment comprising domains 2 and 3; and (b) determining whether the substance is capable of blocking EF-Tu production.

This procedure can be applied both in vitro and in vivo. In an in vitro method, e.g. pure EF-Tu molecules respectively. their suitable fragments are incubated under established conditions under which fibril formation may occur. The fibril forming effect of the test substance can be determined in a simple manner, e.g. immunostaining with a labeled EF-Tu antibody or using EF-Tu molecules having labeled moieties e.g. fluorescent. Of course, the method can also be carried out in vivo, wherein the effect of adding the test substance to the fibril network in the cell can be determined by immunological methods, e.g. immunohistochemically with labeled anti-Tu antibodies and microscopic evaluation.

Agents which block the formation of EF-Tu polymers and which can be obtained by the above process as well as substances derived therefrom e.g. by empirical derivation and / or computer modeling may be formulated as pharmaceutical preparations, optionally together with pharmaceutically acceptable carriers, excipients and / or diluents.

The pharmaceutical composition may be e.g. in the form of a liquid composition, a solid composition, an emulsion or a dispersion. Depending on the formulation, administration may be by injection, oral, rectal, nasal, topical. The dosage, depending on the active ingredient, the form of administration and the nature and severity of the disease, is selected in such a way that the control of the bacterial infection is possible.

The action of the antibacterial agent may vary. Substances that can directly bind to the fusion sites of domains 2 and / or 3 of EF-Tu can be used once. Alternatively, agents that bind to other sites of EF-Tu molecules but have an inhibitory effect on fit and thus result in reduced fibril formation may also be used.

In a preferred embodiment of the invention, peptide antibacterial agents are used. The peptide composition is based on oligopeptides that bind to EFT preferably in the region of the fusion sites of domain 2 and / or 3. These oligopeptides may

Contain the amino acid sequences of domains 2 and / or 3 having a length of preferably 4 to 20 amino acids, particularly preferably 5 to 15 amino acids, and most preferably 6 to 12 amino acids. These sub-regions are capable of binding to the complementary sequences of just the second domain, i. j. sequences from domain 2 are capable of binding to domain 3 and sequences from domain 3 are capable of binding to domain 2.

In another preferred embodiment, the substances that bind to EF-Tu comprise a portion of amino acid sequences from domain 2 having a length of at least 4, and in particular at least 5 amino acids, particularly a portion of the 2 region of amino acids 218 and 224, corresponds to amino acid region 317 to 328 and / or amino acid region 343 to 354 of domain 3 of EF-Tu. Alternatively, substances that comprise sub-regions of amino acid sequences from domain 3 having a length of at least 4 amino acids, in particular at least 5 amino acids, most preferably at least 6 amino acids, and do not compromise any portions corresponding to amino acids 218 to 224 of domain 2 may be. Truncated EF-Tu, consisting exclusively of domain 3 without domains 1 and 2 or exclusively of domains 1 and 2 without domains

3. Such an EF-Tu fragment competes in a cell with cell-synthesized native natural EF-Tu molecules and, when incorporated into the polymerized protofilament, participates in chain fragmentation as there is always a lack of a suitable chain-extending domain. This will no longer create an intact network system. This means the same as loss of viability of the bacterial cell. Disturbance of the network system in the bacterial cell negatively affects the shape and behavior of the bacterial cell, as has been shown in experiments. A negative effect on the shape and behavior of the cell is evidence of the expected cell death that will occur with the antibiotic of the invention.

Instead of the described " truncated " EF-Tu fragment, an antibiotic can also be used to prevent the polymerization of EF-Tu protein molecules, i.e. chain fragmentation is achieved by another means, e.g. by means of partial regions that prevent binding of other EF-Tu protein molecules.

The particular importance of the antibiotics according to the invention is that the risk of developing resistance to this new class of antibiotics is very rare. Resistance by

It meant that the bacterium would break down the peptide introduced into the cell. If this were to happen, the bacterium could not be prevented from degrading its structurally identical peptide, which is part of the EF-Tu own cell and which is of great importance for translation.

The antibacterial compositions may comprise linear or cyclic peptide compounds or peptidomimetics. The peptide compounds may consist of natural L-α-amino acids, sometimes other amino acids, e.g. D-α-amino acids, azaaminide amino acids, β-amino acids, non-genetically encoded L- or D-α-amino acids, etc., or combinations thereof. The production of peptidomimetics is described e.g. in Ripka, A. S., Rich, D. H. (1998), Peptidomimetic Design, Curr. Op. Chem. Biol., 2, 441-452.

The peptide compounds may also contain peptide compounds or peptidomimetic-linked hydrophobic moieties that facilitate cytoplasmic membrane transfer or larger space filling moieties that prevent the deposition of additional EF-Tu molecules and thereby the formation of polymerization products. In addition, the antibacterial agents may have degradation-protecting groups.

Antibacterial agents may be used against any of the prokaryotic organisms of the arachaea, especially pathogenic organisms. Both gram-positive bacteria, gram-negative bacteria and mycoplasmas have an EF-Tu-based cytoskeleton, and therefore the composition of the invention can be used against them. Antibacterial agents may e.g. used against vancomycin-resistant bacteria, e.g. against staphylococci.

The new class of antibiotics thus has a wide range of uses. It has been shown that those regions that are capable of binding monomers to protofilament formation are very similar in all bacteria of interest due to their amino acid sequence. EFT is preserved in this area. Also, the spacing of these regions in a given EF-Tu molecule, i.e. the spacing of the exposed regions of domains 2 and 3, are identical, as expressed by the number of amino acids, with 126 amino acids between the conserved regions.

Antibiotics according to the invention are characterized by a high specificity and in particular a great rarity of adverse effects. Large EF-Tu sequences are in human

-7 cells except mitochondria do not occur. Mytochondrial EF-Tu-like sequences are highly protected from the antibiotic through a double membrane.

The invention is further illustrated by the figures and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the macromolecular architecture of EF-Tu bacterial proteins, in which domains 1, 2 and 3 are described in more detail. This bacterial EFT protein can be deposited during polymerization along with regularly formed fibrils, as shown in FIG. Second

Fig. 3 shows a schematic embodiment of the polymerization, with + or s - designation of the reactive binding regions of domains 2 and 3.

Fig. 4 shows an enlargement (magnification of approximately 1.5 million times) from an electron-microscopic image of an in vivo polymerized isolated fibril from EFT-protein molecules. The upper half of the dotted line represents domains 1, the lower half of superimposed domains 2 and 3.

If the polymerization of the EF-Tu protein in FIG. 3 with + and - labeled binding regions by adding an excess of particles containing the partial regions of the amino acid sequence of domains 2 or 3 will decrease, survival of the affected bacterial cells becomes impossible as cell structure breaks down.

DETAILED DESCRIPTION OF THE INVENTION

Experiments have been carried out with Gram-positive bacterium Thermoanaerobacterium thermosaccharolyticum EM1 (abbreviated as EM1 below) and Mycoplasma pneumoniae cell wall bacterium (abbreviated as Mp below) to demonstrate that these bacteria have a sustained EF-Tu-based cytoskeleton.

The experiments include the identification and cellular localization of candidate proteins for such a bacterial cytoskeleton by using anti-actin antibodies (made against higher cell actin) whose weaker or stronger cross-reactivity with bacterial proteins indicates that the bacteria possess recognizable proteins belonging to the bacterial cytoskeleton. actin supergroup without

-8 showed striking sequence homology with higher cell actin. Prokaryotes do not explicitly have an actin gene.

In addition to immunoelectron microscopy with the above antibodies on ultra-thin sections of bacteria, whole mount techniques were used. By combining these techniques, it has been found that a protein fibril network system is present near the surface of the cytoplasmic membrane facing the cytoplasm and through the cytoplasm, the components of which cross-react with antiactin antibodies. The cytoplasmic membrane and the peripheral part of this network system form two concentric empty tubes, the cytoplasmic membrane being the outer of the two tubes, the peripheral part of the network system (cytoskeleton) internal. The fibrils passing through the cytoplasm complement and stabilize the system and are the site of action of the ribosomes. Ribosomes are also directed towards the cytoplasm on the peripheral part of the cytoskeleton.

Using a French press, EM1 cells were disrupted and SDS gel electrophoresis and Western blotting was performed on the obtained material (soluble fraction, particle fraction). Several defined bands were obtained in the SDS gel, one of which (at approximately 43 kDa) was stainable with anti-actin antibodies as well as anti-EF-Tu antibodies obtained against EF-Tu from Mp. This band was amplified where the particle fraction of cell disruption obtained by low-speed centrifugation was used as material for SDS gel electrophoresis. Anti-EF-Tu antibodies were used because, at 43 kDa, EF-Tu is usually found (representing up to 9% of the protein mass of one bacterium), EF-Tu belongs to the actin supergroup, and EF-Tu is abundant in prokaryotic cells .

The role of EF-Tu as a building block of bacterial cytoskeleton is new. From this feature of EF-Tu as a building block for a complex network system, such as a cytoskeleton, it can be concluded that a bacterial cell has to deploy large amounts of proteins there. A comparison of the construction of such a bacterial cytoskeleton with higher cells makes it clear that a bacterial cytoskeleton could also be made from several protein types. EF-Tu is the main ingredient. In higher cells, although EF-Tu is not involved in the production of cytoskeleton (the higher cell has no EFT), it is known that many different proteins are involved in cytoskeleton.

It was shown that the components of the above-described cytoskeleton network system, reacting with anti-actin antibodies in sections and whole mount, also reacted intensively with anti-EF-Tu antibodies.

This reaction was carried out with Mp on a total, tritone treatment (removal of the cytoplasmic membrane) released now towards the initially covered surfaces exposed to the environment. A control made using cells that were not triton-treated but otherwise treated and did not lose the cytoplasmic membrane showed no labeling. Thus, in this controlled experiment, the cytoplasmic membrane concealed potential binding sites for EF-Tu.

The surface released after removal of the cytoplasmic membrane is a peripheral part of the cell's cytoskeleton. In this situation, the internal components of the cell, such as e.g. ribosomes that have been shown to be the attachment sites of the EF-Tu performing the helper function during translation (EF-Tu functions here through its domain 1) during translation. From this, it was concluded that during translation, the EF-Tu cribosome does not arrive, but the ribosome to EF-Tu, which by its property of the cytoskeleton component is spatially fixed on the periphery of the cell and on the transverse fibrils in the cytoplasm.

The presence of persistent bacterial cytoskeleton was further demonstrated in the eubacteria of Escherichia coli, Bacillus sp., Ralstonia eutropha and Thermoanaerobacterium thermosulfurigenes, as well as in Archaea Methanococcus jannaschii and methanococcus voltae.

Claims (22)

PATENT CLAIMS Use of substances bound to EF-Tu to prevent the formation of cytoskeletons in bacterial cells. Use according to claim 1, wherein the substances bind to EF-Tu in the region of domain 2 (amino acids 205 to 298) and / or domain 3 (amino acids 299 to 394). Use according to claim 1 or 2, wherein the substances bind to EF-Tu in the region of amino acids 218 to 224 of domain 2. Use according to claim 1 or 2, wherein the substances bind to EF-Tu in the region of amino acids 317 to 328 and / or 343 to 354 of domain 3. Use according to claims 1 to 4, wherein the substances comprise partial stretches of amino acid sequences from domains 2 and / or 3 having a length of 4 to 20 amino acids. The use of claim 5, wherein the amino acid sequence sub-regions are 6 to 12 amino acids in length. The use according to claims 1 to 6, wherein the substances are selected from linear or cyclic peptide compounds or from peptidomimetics. The use according to claim 7, wherein the hydrophobic grouped peptide compounds or peptidomimetics comprise a larger space occupation group and / or a group with degradation protective functions. Use according to any one of claims 1 to 8 for the manufacture of an antibacterial composition. -11 Use according to claim 9, wherein the antibacterial composition is in the form of a pharmaceutical composition, optionally together with pharmaceutically acceptable carriers, diluents and / or excipients. Use according to claim 9 or 10 for the manufacture of a composition against Gram-positive or Gram-negative bacteria. Use according to claim 9 or 10 for the production of an anti-mycoplasma agent. 13. An antibacterial composition comprising partial stretches of amino acid sequences of domain 2 and / or domain 3 of EF-Tu bacterial proteins having a length of 4 to 20 amino acids. 14. Antibacterial composition according to claim 13, characterized in that it comprises sub-regions of 5 to 15 amino acids in length. 15. Antibacterial composition according to claim 13 or 14, characterized in that it comprises sub-regions of 6 to 12 amino acids in length. Antibacterial composition according to claims 13 to 15, characterized in that the hydrophobic groupings grouped by peptide compounds or peptidomimetics comprise a group with greater space occupancy and / or groups with protective functions against degradation. Antibacterial composition according to claims 13 to 16, characterized in that it is in the form of a pharmaceutical preparation, optionally together with pharmaceutically acceptable carriers, diluents and / or excipients. 18. A method for identifying novel antibacterial active substances comprising: 12 (a) contacting one of the test substances with a bacterial EF-Tu or a fragment thereof capable of polymerizing, e.g. with a fragment comprising domains 2 and 3, and (b) determining whether the substance can block the formation of EF-Tu polymers. The method according to claim 18, characterized in that it is carried out as an in vitro test. 20. The method of claim 18, wherein the method is an in vivo test. Method according to claim 18, characterized in that labeled EF-Tu proteins or polymerizable partial fragments thereof are used to determine the formation of EF-Tu polymers. Process according to any one of claims 18 to 21, characterized in that the substance which inhibits the formation of EF-Tu polymers or a substance derived therefrom is formulated as a pharmaceutical composition, optionally together with pharmaceutically acceptable carriers, diluents. and / or excipients.